Docetaxel immunoassay

ABSTRACT

Novel conjugates of docetaxel and novel docetaxel immunogens derived from the 7 and 10 positions of docetaxel and monoclonal antibodies generated by these docetaxel linked immunogens are useful in immunoassays for the quantification and monitoring of docetaxel in biological fluids.

FIELD OF THE INVENTION

This invention relates to the field of immunological assays fordetermining the presence and/or quantifying the amount of docetaxel inhuman biological fluids in order to rapidly determine optimal drugconcentrations during chemotherapy.

BACKGROUND OF THE INVENTION

Cancer is a term used to describe a group of malignancies that all sharethe common trait of developing when cells in a part of the body begin togrow out of control. Most cancers form as tumors, but can also manifestin the blood and circulate through other tissues where they grow. Cancermalignancies are most commonly treated with a combination of surgery,chemotherapy, and/or radiation therapy. The type of treatment used totreat a specific cancer depends upon several factors including the typeof cancer malignancy and the stage during which it was diagnosed.

Taxotere, whose chemical name is docetaxel, is one of the more commoncytotoxic agents used for the treatment of Breast (Holmes et. al. Proc.Am. Soc. Clin. Oncol., 10, 60, 1991), Ovarian (Einzig et. al. Proc. Am.Assoc. Cancer Res., 31, 1114, 1990) and non-small cell lung cancer.Docetaxel, which is also known as Taxotere, has the formula:

This compound has been associated with debilitating side effects such asbone marrow density loss, allergic reaction, neutropenia, hypotension,bardycardia, nausea and vomiting. By monitoring the levels of docetaxelin the body and adjusting the dose these side effects can be bettercontrolled and limited in patients.

At the same time, there is often highly variable relationship betweenthe dose of docetaxel and the resulting serum drug concentration thataffects therapeutic effect. The degree of intra- and inter-individualpharmacokinetic variability of docetaxel can be as high as 5-fold(Gurney et. al., J. Clin. Oncol. 14, pp 2590-2611, 1996) and is impactedby many factors, including:

Organ function

Genetic regulation

Disease state

Age

Drug-drug interaction

Time of drug ingestion,

Mode of drug administration

Technique-related administration

As a result of this variability, equal doses of the same drug indifferent individuals can result in dramatically different clinicaloutcomes (Hon et. al. Clinical Chemistry 44, pp 388-400, 1998). Theeffectiveness of the same docetaxel dosage varies significantly basedupon individual drug clearance and the ultimate serum drug concentrationin the patient. Therapeutic drug management would provide the clinicianwith insight on patient variation in both oral and intravenous drugadministration. With therapeutic drug management, drug dosages could beindividualized to the patient, and the chances of effectively treatingthe cancer, without the unwanted side effects, would be much higher.

In addition, therapeutic drug management of docetaxel would serve as anexcellent tool to ensure compliance in administering chemotherapy withthe actual prescribed dosage and achievement of the effective serumconcentration levels. It has been found that variability in serumconcentration is not only due to physiological factors, but can alsoresult from variation in administration technique.

Routine therapeutic drug management of docetaxel would require theavailability of simple automated tests adaptable to general laboratoryequipment. Tests that best fit these criteria are immunoassays. In orderto be an effective immunoassay the antibody should be most specific forthe active form of the drug and display very low cross-reactivity forthe inactive forms of the drug. Currently there are no availableimmunoassays available for determining levels of docetaxel in plasma orblood.

SUMMARY OF INVENTION

In accordance with this invention, a new class of antibodies have beenproduced which are substantially selectively reactive to docetaxel so asto bind to docetaxel without any substantial cross reactivity topaclitaxel and related compounds. By selectively reactive it is meantthat this antibody reacts with docetaxel and does not substantiallyreact with docetaxel metabolites and docetaxel related compounds such astaxol or 10-O-Deacetylbaccatin III.

It has been found that by using immunogens which are conjugates of animmunogenic polyamine polymer with a ligand selected from the groupconsisting of a 10-hydroxydocetaxel derivatives of the formula:

7-hydroxydocetaxel derivatives of the formula:

7,10-dihydroxy docetaxel derivatives of the formula:

wherein B is —CH₂—;

Y is an organic spacing group;

X is a terminal functional group capable of binding to a carrier;

p is an integer from 0 to 1; and

mixtures thereof; produce antibodies which are specific for docetaxeland do not substantially react with or bind to taxol and metabolites orrelated compounds of docetaxel. The provision of these antibodies whichsubstantially selectively react with docetaxel and do not cross reactwith taxol or metabolites of docetaxel allows one to produce animmunoassay which can specifically detect and monitor docetaxel in thefluid samples of patients being treated with docetaxel. Also includedwithin this invention are reagents and kits for said immunoassay.

DETAILED DESCRIPTION

In accordance with this invention, a new class of antibodies is providedwhich substantially selectively reacts with docetaxel and do notsubstantially react or cross react with docetaxel like compounds such astaxol and 10-O-deacetylbaccatin III. It has been discovered that throughthe use of these docetaxel derivatives of formula II-A, II-B and II-C ormixtures thereof; as immunogens, this new class of antibodies of thisinvention are provided. It is through the use of these antibodies thatan immunoassay, including reagents and kits for such immunoassay fordetecting and/or quantifying docetaxel in blood, plasma or other bodyfluid samples has been developed. By use of this immunoassay, thepresence and amount of docetaxel in body fluid samples, preferable ablood or plasma sample, can be detected and/or quantified. In thismanner, a patient being treated with docetaxel, can be monitored duringtherapy and treatment adjusted in accordance with said monitoring. Bymeans of this invention one achieves the therapeutic drug management ofdocetaxel in cancer patients being treated with docetaxel as achemotherapeutic agent.

The reagents utilized in the assay of this invention are conjugates of acarrier, preferably containing polyamine functional groups, with thecompounds of formula II-A, II-B and II-C or mixtures thereof. Theseconjugates are competitive binding partners with the docetaxel presentin the sample for the binding with the antibodies of this invention.Therefore, the amount of conjugate reagent which binds to the antibodywill be inversely proportional to the amount of docetaxel in the sample.In accordance with this invention, the assay utilizes any conventionalmeasuring means for detecting and measuring the amount of said conjugatewhich is bound or unbound to the antibody. Through the use of saidmeans, the amount of the bound or unbound conjugate can be determined.Generally, the amount of docetaxel in a sample is determined bycorrelating the measured amount of the bound or unbound conjugateproduced by the docetaxel in the sample with values of the bound orunbound conjugate determined from standard or calibration curve samplescontaining known amounts of docetaxel, which known amounts are in therange expected for the sample to be tested. These studies for producingcalibration curves are determined using the same immunoassay procedureas used for the sample.

The conjugates, as well as the immunogens, are prepared from compoundsof the formula II-A, II-B and II-C or mixtures thereof. In theconjugates or immunogens, the carrier and the polyamine polymer arelinked to ligand portions of the compounds of formula II-A, II-B andII-C. The ligand portions have the formula:

wherein Y, A and p are as above; and

X′ is —CH₂— or a functional linking group;compounds of the formula:

and compounds of the formula:

These ligand portions may be linked to one or more active sites on thecarrier of the conjugate or polyamine polymer of the immunogen. When thecompounds of formula II-A, II-B or II-C are used to make immunogens, Xin the compound of formula II-A, II-B and II-C and X¹ in the compound offormula III-A, III-B and III-C are X₂ or X₂ ¹ which are functionalgroups capable of binding or linking to a polyamine polymer.

Definitions

Throughout this description the following definitions are to beunderstood: The term “alkylene” designates a divalent saturated straightor branch chain hydrocarbon substituent containing from one to tencarbon atoms

The terms “immunogen” and “immunogenic” refer to substances capable ofeliciting, producing, or generating an immune response in an organism.

The term “conjugate” refers to any substance formed from the joiningtogether of two parts. Representative conjugates in accordance with thepresent invention include those formed by the joining together of asmall molecule, such as the compound of formula II-A, II-B and II-C anda large molecule, such as a carrier, preferably carriers which comprisea polyamine polymer, particularly a protein. In the conjugate the smallmolecule maybe joined or linked at one or more active sites on the largemolecule. The term conjugate includes the term immunogen. In theconjugates used as reagents the carrier can be any carrier and X csn beany functional group which can be linked to a carrier. In the immunogenthe carrier is a polyamine polymer and X is any functional group capableof linling to a polyamine polymer.

“Haptens” are partial or incomplete antigens. They are protein-freesubstances, mostly low molecular weight substances, which are notcapable of stimulating antibody formation, but which do react withantibodies. The latter are formed by coupling a hapten to a highmolecular weight immunogenic carrier and then injecting this coupledproduct, i.e., immunogen, into a human or animal subject. The hapten ofthis invention is docetaxel.

As used herein, a “spacing group” or “spacer” refers to a portion of achemical structure which connects two or more substructures such ashaptens, carriers, immunogens, labels, or tracer through a CH₂ orfunctional linking group. These spacer groups will be enumeratedhereinafter in this application. The atoms of a spacing group and theatoms of a chain within the spacing group are themselves connected bychemical bonds.

Among the preferred spacers are straight or branched, saturated orunsaturated, carbon chains. Theses carbon chains may also include one ormore heteroatoms within the chain or at termini of the chains. By“heteroatoms” is meant atoms other than carbon which are chosen from thegroup consisting of oxygen, nitrogen and sulfur. Spacing groups may alsoinclude cyclic or aromatic groups as part of the chain or as asubstitution on one of the atoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. A functional linking group may be used to activate, e.g.,provide an available functional site on, a hapten or spacing group forsynthesizing a conjugate of a hapten with a label or carrier orpolyamine polymer.

An “immunogenic carrier,” as the terms are used herein, is animmunogenic substance, commonly a protein, that can join with a hapten,in this case docetaxel or the docetaxel derivatives hereinbeforedescribed, thereby enabling these hapten derivatives to induce an immuneresponse and elicit the production of antibodies that can bindspecifically with these haptens. The immunogenic carriers and thelinking groups will be enumerated hereinafter in this application. Amongthe immunogenic carrier substances are included proteins, glycoproteins,complex polyamino-polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host. The polyamino-polysaccharides may be prepared frompolysaccharides using any of the conventional means known for thispreparation.

Also various protein types may be employed as a poly(amino acid)immunogenic carrier. These types include albumins, serum proteins,lipoproteins, etc. Illustrative proteins include bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin, bovinethyroglobulin (BTG) etc. Alternatively, synthetic poly(amino acids) maybe utilized.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymers built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

“Poly(amino acid)” or “polypeptide” is a polyamide formed from aminoacids.

Poly(amino acids) will generally range from about 2,000 molecularweight, having no upper molecular weight limit, normally being less than10,000,000 and usually not more than about 600,000 daltons. There willusually be different ranges, depending on whether an immunogenic carrieror an enzyme is involved.

A “peptide” is any compound formed by the linkage of two or more aminoacids by amide (peptide) bonds, usually a polymer of α-amino acids inwhich the α-amino group of each amino acid residue (except the NH₂terminus) is linked to the α-carboxyl group of the next residue in alinear chain. The terms peptide, polypeptide and poly(amino acid) areused synonymously herein to refer to this class of compounds withoutrestriction as to size. The largest members of this class are referredto as proteins.

A “label,” “detector molecule,” or “tracer” is any molecule whichproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, immunogen, antibody, or to anothermolecule such as a receptor or a molecule that can bind to a receptorsuch as a ligand, particularly a hapten. Non-limiting examples of labelsinclude radioactive isotopes, enzymes, enzyme fragments, enzymesubstrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,chemiluminescers, luminescers, or sensitizers; a non-magnetic ormagnetic particle, a solid support, a liposome, a ligand, or a receptor.

The term “antibody” refers to a specific protein binding partner for anantigen and is any substance, or group of substances, which has aspecific binding affinity for an antigen to the exclusion of othersubstances. The generic term antibody subsumes polyclonal antibodies,monoclonal antibodies and antibody fragments.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “carrier” refers to solid particles and/or polymeric polymerssuch as immunogenic polymers such as those mentioned above. Where thecarrier is a solid particle, the solid particle may be bound, coatedwith or otherwise attached to the polymeric material which preferably isa polyamine polymer to provide one or more reactive sites for bonding tothe terminal functional group X in the compounds of the formula II-A,II-B and II-C.

The term “reagent kit,” or “test kit,” refers to an assembly ofmaterials that are used in performing an assay. The reagents can beprovided in packaged combination in the same or in separate containers,depending on their cross-reactivities and stabilities, and in liquid orin lyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. A reagent kit embodying features of the presentinvention comprises antibodies specific for docetaxel. The kit mayfurther comprise ligands of the analyte and calibration and controlmaterials. The reagents may remain in liquid form or may be lyophilized.

The phrase “calibration and control materials” refers to any standard orreference material containing a known amount of a drug to be measured.The concentration of drug is calculated by comparing the resultsobtained for the unknown specimen with the results obtained for thestandard. This is commonly done by constructing a calibration curve.

The term “biological sample” includes, but is not limited to, anyquantity of a substance from a living thing or formerly living thing.Such living things include, but are not limited to, humans, mice,monkeys, rats, rabbits, horses, and other animals. Such substancesinclude, but are not limited to, blood, serum, plasma, urine, cells,organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,chondrocytes, synovial macrophages, endothelial cells, and skin.

Reagents and Immunogens

In constructing an immunoassay, a conjugate of docetaxel is constructedto compete with the docetaxel in the sample for binding sites on theantibodies. In the immunoassay of this invention, the reagents areconjugates of a carrier with a) the 10-substituted docetaxel derivativesof the compounds of formula II-A; b) the 7-docetaxel derivatives offormula II-B and c) the 7,10-disubstituted derivatives of docetaxel offormula II-C or mixtures thereof. In the compounds of formula III-A,III-B and III-C, the linker spacer constitutes the —CH₂—(Y)_(p) X′—portion of this molecule. These linker X′ and the spacer —CH₂—(Y)_(p)—are conventional in preparing conjugates and immunogens.

Any of the conventional spacer-linking groups utilized to prepareconjugates and immunogens for immunoassays can be utilized in thecompounds of formula III-A, III-B and III-C. Such conventional linkersand spacers are disclosed in U.S. Pat. No. 5,501,987 and U.S. Pat. No.5,101,015.

Among the preferred spacer groups are included the spacer groupshereinbefore mentioned. Particularly preferred spacing groups are groupssuch as alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6 with alkylene being the especially preferred spacing group.

In the compounds of formula III-A, III-B and III-C, X′ is —CH₂— or afunctional group linking the spacer to the carrier, preferably to anamine group on the polymeric carrier. The group X′ is the result of theterminal functional group X in the compounds of Formula II-A, II-B andII-C which is capable of binding to a carrier, preferably to an aminogroup in the polyamine polymer present in the carrier or used as theimmunogen. Any terminal functional group capable of binding to acarrier, preferably capable of reacting with an amine can be utilized asthe functional group X in the compounds of formula II-A, II-B, and II-C.These terminal functional groups preferably included within X are:

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur. The radical —N═C═R₄,can be an isocyanate or an isothiocyanate. The active esters formed byOR₃ include imidoester, such as N-hydroxysuccinamide, 1-hydroxybenzotriazole and p-nitrophenyl ester. However any active ester whichcan react with an amine group can be used.

The carboxylic group and the active esters are coupled to the carrier orimmunogenic polymer by conventional means. The amine group on thepolyamine polymer, such as a protein, produces an amide group whichconnects the spacer to the polymer, immunogens or carrier and/orconjugates of this invention. On the other hand, carriers can be coatedwith a polyamine polymer to supply the amino group for linking to theligand portion.

In the immunogens and conjugates of the present invention, the chemicalbonds between the carboxyl group-containing docetaxel haptens and theamino groups on the polyamine polymer on the carrier or immunogen can beestablished using a variety of methods known to one skilled in the art.It is frequently preferable to form amide bonds. Amide bonds are formedby first activating the carboxylic acid moiety of the docetaxel haptenin the compounds of formula II-A, II-B and II-C by reacting the carboxygroup with a leaving group reagent (e.g., N-hydroxysuccinimide,1-hydroxybenzotriazole, p-nitrophenol and the like). An activatingreagent such as dicyclohexylcarbodiimide, diisopropylcarbodiimide andthe like can be used. The activated form of the carboxyl group in thedocetaxel hapten of formula II-A, II-B and II-C is then reacted with abuffered solution containing the protein carrier.

In cases where the docetaxel derivative of formula II-A, II-B and II-Ccontains a primary or secondary amino group as well as the carboxylgroup, it is necessary to use an amine protecting group during theactivation and coupling reactions to prevent the conjugates fromreacting with themselves. Typically, the amines on the conjugate areprotected by forming the corresponding N-trifluoroacetamide,N-tertbutyloxycarbonyl urethane (N-t-BOC urethane), N-carbobenzyloxyurethane or similar structure. Once the coupling reaction to theimmunogenic polymer or carrier has been accomplished, as describedabove, the amine protecting group can be removed using reagents that donot otherwise alter the structure of the immunogen or conjugate. Suchreagents and methods are known to one skilled in the art and includeweak or strong aqueous or anhydrous acids, weak or strong aqueous oranhydrous bases, hydride-containing reagents such as sodium borohydrideor sodium cyanoborohydride and catalytic hydrogenation. Various methodsof conjugating haptens and carriers are also disclosed in U.S. Pat. No.3,996,344 and U.S. Pat. No. 4,016,146, which are herein incorporated byreference.

On the other hand where X is a terminal isocyanide or isothiocyanateradical in the compound of formula II-A, II-B and II-C, these radicalswhen reacted with the free amine of a polyamine polymer produce theconjugate or the immunogen where X′ is,

in the ligand portions of formula III-A, III-B and III-C, functionallyconnects with the amino group on the polyamine containing carrier or theimmunogenic polypeptide.

Where X, in the compounds of formula II-A, II-B and II-C, is an aldehydegroup these compounds may be connected to the amine group of thepolyamine polypeptide or carrier through an amine linkage by reductiveamination. Any conventional method of condensing an aldehyde with anamine such as through reductive amination can be used to form thislinkage. In this case, X′ in the ligand portions of formula III-A, III-Band III-C is —CH₂—.

In preparing the 7,10-monoderivatives of formula II-A and II-B and the7,10-di substituted derivatives of docetaxel, the 2′-hydroxy group ofdocetaxel is first protected.

This 2′-hydroxy group is on the side chain extending from the13-position on the docetaxel ring structure. This is the most reactiveof the hydroxy groups in docetaxel.

Any conventional method of protecting a hydroxy group such as by anesterification can be utilized to protect this hydroxy group at the 2′position, while leaving the hydroxy groups at the 7 and 10 positionsfree for reaction. Any of the conventional hydroxy protecting groups canbe utilized to accomplish this purpose. A preferred hydroxy protectinggroup is the allylorthoformate ester group which is formed by reactingthe compound of formula I with allylchloroformate by conventional meanswell known in the art. This is an easily produced protecting group whichcan be easily removed at a later stage in the process.

After protecting the 2′ hydroxy group, this protected docetaxel offormula I can be converted into the 10-docetaxel derivative of formulaII-A, the 7-docetaxel derivative of formula II-B or the 7,10-docetaxelderivative of formula II-C depending upon the molar quantity of reagentsutilized to react with the 2′ protected docetaxel of formula I. Ingeneral, where a molar excess of the reagent is reacted with the 2′protected docetaxel of formula I, the resulting final product will be amixture of the 7-0 and 10-0 substituted derivatives, as well as the7,10-0 disubstituted derivatives. These derivatives can be separatedusing a silica gel column and a gradient comprising dichloromethane andethyl acetate, generally 100% dichloromethane at the start whilegradually adding ethyl acetate to the column. The individual ingredientscan be collected and their structure confirmed by NMR.

In carrying out this reaction the 7 hydroxy group in the 2′ hydroxyprotected docetaxel will react first with the reagent such as thecompound of formula V-A. Therefore, by limiting the ratio of the reagentsuch as the compound of formula V-A or VI which is reacted with thecompound of formula I to about 0.9 to 1.5 moles per mole, the finalproduct will substantially consist of the compounds of formula II-B.Increasing the mole ratio of the reagents reacted with the 2′ protectedhydroxy docetaxel of formula I will produce more of the compounds offormula II-A and II-C in the product. These derivatives can be separatedfrom the product as described above.

The 10 and 7-substituted derivatives of formula II-A and II-B where B is—CH₂—, as well as the 7,10-disubstituted derivatives of formula II-C areformed by reacting the 7 and 10-hydroxy group of docetaxel with a halideof the formula:halo-CH₂—(Y)_(p)—X  V-A

wherein p, Y and X are as above.

In forming these derivatives, any conventional means of reacting analcohol to form an ether can be utilized in condensing the compound offormula V-A with the 7-hydroxy position on the docetaxel. The use of ahalide in the compound of formula V-A provides an efficient means forforming an ether by condensing with the alcohol. On the other hand,where the compound of formula V-A contains functional groups, which mayinterfere with this reaction to form these derivatives, these functionalgroups can be protected by means of suitable protecting groups which canbe removed after this reaction as described hereinabove.

The above derivatives of formula II-A, II-B or II-C where B is

are produced by reacting one or more of the free hydroxy groups on the2′ protected docetaxel with an amino compound of the formula:NH—CH₂—(Y)_(p)—X  VI

wherein X, Y and p are as above,after first converting the one or more hydroxy groups on the 2′protected docetaxel to the chloroformate group

Any conventional means of converting a hydroxy group to a chloroformategroup can be used. After the formulation of a chloroformate, the halogroup of the chloroformate is condensed with the amine group in thecompound of formula VI. Prior to this reaction, the reactive group ondocetaxel and/or on the compound of formula VI are protected asdescribed hereinabove with a conventional protecting group. Theseprotecting groups can be removed after this halide condensation byconventional means such as described hereinbefore.

The compounds of formula II-A, II-B and II-C can be converted into theimmunogens and/or the conjugate reagents of this invention by reactingthese compounds with a carrier, preferably a polyamine polypeptide or acarrier coated with a polyamine polypeptide. The same polypeptide can beutilized as the carrier and as the immunogenic polymer in the immunogenof this invention provided that polyamines or polypeptides areimmunologically active. However, to form the conjugates used as reagentsin the immunoassay, these polymers need not produce an immunologicalresponse as needed for the immunogens. In accordance with thisinvention, the various functional group represented by X in thecompounds of formula II-A, II-B and II-C can be conjugated to thecarrier by conventional means of attaching a functional group to acarrier. In accordance with a preferred embodiment, in the compounds offormula II-A, II-B and II-C, X is a carboxylic acid group.

Antibodies

The present invention also relates to novel antibodies includingmonoclonal antibodies to docetaxel produced by utilizing theaforementioned immunogens. In accordance with this invention it has beenfound that these antibodies produced in accordance with this inventionare selectively reactive with docetaxel and do not react with taxol ordocetaxel or with metabolites of docetaxel derivatives which wouldinterfere with immunoassays for docetaxel. The most problematic of theseis taxol, whose chemical name is paclitaxel. The ability of theantibodies of this invention not to react with the taxol and taxolmetabolites make these antibodies particularly valuable in providing animmunoassay for docetaxel. In addition, these antibodies do not reactwith related taxol or docetaxel like compounds such as10-O-Deacetylbaccatin III which contain the docetaxel or taxol ringstructure, as well as with other docetaxel metabolites or analogs,except analogs derived from the 7-hydroxy or 10-hydroxy derivatives ofthe compound of formula II-A, II-B and II-C. 10-O-Deacetylbaccatin IIIhas the formula:

The present invention relates to novel antibodies and monoclonalantibodies to docetaxel. The antisera of the invention can beconveniently produced by immunizing host animals with the immunogens ofthis invention. Suitable host animals include rodents, such as, forexample, mice, rats, rabbits, guinea pigs and the like, or highermammals such as goats, sheep, horses and the like. Initial doses,bleedings and booster shots can be given according to accepted protocolsfor eliciting immune responses in animals. Through periodic bleeding,the blood samples of the immunized mice were observed to develop animmune response against docetaxel binding utilizing conventionalimmunoassays. These methods provide a convenient way to screen for hostsand antibodies which are producing antisera having the desired activity.The antibodies were also screened against taxol and antibodies wereproduced which showed no substantial binding to taxol.

Monoclonal antibodies are produced conveniently by immunizing Balb/cmice according to the schedule followed by injecting the mice withadditional immunogen i.p. or i.v. on three successive days startingthree days prior to the cell fusion. Other protocols well known in theantibody art may of course be utilized as well. The completeimmunization protocol detailed herein provided an optimum protocol forserum antibody response for the antibody to docetaxel.

B lymphocytes obtained from the spleen, peripheral blood, lymph nodes orother tissue of the host may be used as the monoclonal antibodyproducing cell. Most preferred are B lymphocytes obtained from thespleen. Hybridomas capable of generating the desired monoclonalantibodies of the invention are obtained by fusing such B lymphocyteswith an immortal cell line, which is a cell line that which imparts longterm tissue culture stability on the hybrid cell. In the preferredembodiment of the invention the immortal cell may be a lymphoblastoidcell or a plasmacytoma cell such as a myeloma cell, itself an antibodyproducing cell but also malignant. Murine hybridomas which producedocetaxel monoclonal antibodies are formed by the fusion of mousemyeloma cells and spleen cells from mice immunized with theaforementioned immunogenic conjugates. Chimeric and humanized monoclonalantibodies can be produced by cloning the antibody expressing genes fromthe hybridoma cells and employing recombinant DNA methods now well knownin the art to either join the subsequence of the mouse variable regionto human constant regions or to combine human framework regions withcomplementary determining regions (CDR's) from a donor mouse or ratimmunoglobulin. An improved method for carrying out humanization ofmurine monoclonal antibodies which provides antibodies of enhancedaffinities is set forth in International Patent Application WO 92/11018.

Polypeptide fragments comprising only a portion of the primary antibodystructure may be produced, which fragments possess one or moreimmunoglobulin activities. These polypeptide fragments may be producedby proteolytic cleavage of intact antibodies by methods well known inthe art, or by inserting stop codons at the desired locations inexpression vectors containing the antibody genes using site-directedmutageneses to produce Fab fragments or (Fab′)₂ fragments. Single chainantibodies may be produced by joining VL and VH regions with a DNAlinker (see Huston et al., Proc. Natl. Acad. Sci. U.S., 85:5879-5883(1988) and Bird et al., Science, 242:423-426 (1988))

The antibodies of this invention are selective for docetaxel and do nothave any substantial cross-reactivity with taxol. By having nosubstantial cross-reactivity it is meant that the antibodies of thisinvention have a cross reactivity relative to docetaxel with taxol orthese metabolites of 6% or less.

Immunoassays

In accordance with this invention, the conjugated and the antibodiesgenerated from the immunogens of thse compounds of formula II-A, II-Band II-C or mixtures thereof can be utilized as reagents for thedetermination of docetaxel in patient samples. This determination isperformed by means of an immunoassay. Any immunoassay in which thereagent conjugates formed from the compounds of formula II-A, II-B andII-C compete with the docetaxel in the sample for binding sites on theantibodies generated in accordance with this invention can be utilizedto determine the presence of docetaxel in a patient sample. The mannerfor conducting such an assay for docetaxel in a sample suspected ofcontaining docetaxel, comprises combining an (a) aqueous medium sample,(b) an antibody to docetaxel generated in accordance with this inventionand (c) the conjugates formed from the compounds of formula II-A, II-Band II-C or mixtures thereof. The amount of docetaxel in the sample canbe determined by measuring the inhibition of the binding to the specificantibody of a known amount of the conjugate added to the mixture of thesample and antibody. The result of the inhibition of such binding of theknown amount of conjugates by the unknown sample is compared to theresults obtained in the same assay by utilizing known standard solutionsof docetaxel.

In determining the amount of docetaxel in an unknown sample, the sample,the conjugates formed from the compounds of formula II-A, II-B and II-Cand the antibody may be added in any order.

Various means can be utilized to measure the amount of conjugate formedfrom the compounds of formula II-A, II-B and II-C bound to the antibody.One method is where binding of the conjugates to the antibody causes adecrease in the rate of rotation of a fluorophore conjugate. The amountof decrease in the rate of rotation of a fluorophore conjugate in theliquid mixture can be detected by the fluorescent polarization techniquesuch as disclosed in U.S. Pat. No. 4,269,511 and U.S. Pat. No.4,420,568.

On the other hand, the antibody can be coated or absorbed onnanoparticles so that when these particles react with the docetaxelconjugates formed from the compounds of formula II-A, II-B and II-C,these nanoparticles form an aggregate. However, when the antibody coatedor absorbed nanoparticles react with the docetaxel in the sample, thedocetaxel from the sample bound to these nanoparticles does not causeaggregation of the antibody nanoparticles. The amount of aggregation oragglutination can be measured in the assay mixture by absorbance.

On the other hand, these assays can be carried out by having either theantibody or the docetaxel conjugates attached to a solid support such asa microtiter plate or any other conventional solid support includingsolid particles. Attaching antibodies and proteins to such solidparticles is well known in the art. Any conventional method can beutilized for carrying out such attachments. In many cases, in order toaid measurement, labels may be placed upon the antibodies, conjugates orsolid particles, such as radioactive labels or enzyme labels, as aids indetecting the amount of the conjugates formed from the compounds offormula II-A, II-B and II-C which is bound or unbound with the antibody.Other suitable labels include chromophores, fluorophores, etc.

As a matter of convenience, assay components of the present inventioncan be provided in a kit, a packaged combination with predeterminedamounts of new reagents employed in assaying for docetaxel. Thesereagents include the antibody of this invention, as well as, theconjugates formed from the compounds of formula II-A, II-B and II-C ormixtures thereof. It is generally preferred that in a given immunoassay,if a conjugate formed from a compound of formula II-B is utilized, thatthe antibody be generated by a immunogen formed from a compound offormula II-B. In a like manner, if a conjugate formed from a compound offormula II-B or II-C is utilized, the antibody be generated by theimmunogen formed from the same compound is used for the conjugate.However, this need not be the case and antibodies and conjugates in agiven assay can be derived from any one or of these conjugates andimmunogens. In carrying out an immunoassay in accordance with thisinvention the radicals p, X, Y and B in the reagent and the immunogenwhich forms the antibody used in a given immunoassay can be the same orbe a different substituent within the groups defined for each of thesesradicals. Therefore while the definitions of the radicals p, X, Y, and Bare the same for the conjugate reagent and the immunogen, the particularsubstituent which these radicals represent for the immunogen and theconjugate reagent in a given assay may be different.

In addition to these necessary reagents, additives such as ancillaryreagents may be included, for example, stabilizers, buffers and thelike. The relative amounts of the various reagents may vary widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Reagents can beprovided in solution or as a dry powder, usually lyophilized, includingexcipients which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing the assay.

EXAMPLES

In the examples, the following abbreviations are used for designatingthe following:

-   -   EA Ethyl alcohol    -   MeOH Methanol    -   EtOAc Ethyl acetate    -   DCM Dichloromethane    -   DMAP Dimethylaminopyridine    -   Et₃N Triethyl amine    -   NHS N-hydroxy-succinimide    -   EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride    -   TLC Thin Layer Chromatrography    -   KLH Keyhole Limpet Hemocyanin    -   ANS 8-Anilino-1-naphthalenesulfonic acid    -   i.p. Intraperitoneal    -   HRP Horse radish-peroxidase    -   TMB 3,3′,5,5′-Tetramethylbenzidine    -   TRIS Tris(hydroxymethyl)aminomethane hydrochloride    -   BSA Bovine serum albumin    -   BTG Bovine thyroglobulin    -   PBS Phosphate buffered saline    -   di deionized water

In the examples, Scheme 1 and Scheme 2 below set forth the specificcompounds prepared and referred to by numbers in the Examples. Theschemes are as follows:

Examples Example 1 Preparation of C7, C10 Docetaxel Di-acid Derivative[4] Scheme I

Docetaxel [α](500 mg) was added to a three-neck flask in 20 mL offreshly distilled dichloro methane, under a continuous flow of argon.The temperature was maintained at −15° C., at which timediisopropylethylamine (2 eq.) and allyl chloroformate (1.1 eq.) wasadded. The reaction mixture temperature was brought to room temperatureand allowed to stir for 5 hours. 20 mL of dichloromethane was added andthe mixture was washed with 0.1N HCl (60 mL), dried on Na₂SO₄, andconcentrated on a rotary evaporator. Crude material was purified on asilica gel column with EtOAc/DCM as the gradient (30% EtOAc:71% DCM) toyield [2] (468 mg, 84.78%) as an off-white solid.

To a solution of the alloc-protected docetaxel, [2], (511 mg, 0.57 mmol)and DMAP (0.22 mmol) in DCM (50 mL) under nitrogen, Et₃N (0.22 mmol) wasadded followed by addition of glutaric anhydride (2 eq). The resultingmixture was allowed to stir overnight at room temperature. DCM wasremoved under vacuum and the crude material was purified on a silica gelcolumn with EtOAc/DCM gradient (40% EtOAc:60% DCM) to yield [3] (194 mg,30.23%) as an off-white solid.

Derivative [3] (0.173 mmol) was dissolved in 6 mL of dichloromethaneunder argon and then PhSiH₃ (1.04 mmol) was added along with Pd (PPh₃)₄(0.008 mmol). After 4 hours, 1.5 mL of MeOH was added and the mixturewas stirred for an additional 10 minutes. The reaction mixture wasevaporated to dryness to yield the deprotected docetaxel derivative [4].

Derivative [4] was purified on a silica gel column (60% EtOAc:40% DCM assolvent system) to separate this derivative from the presence of theother derivatives such as the 7-mono docetaxel derivate and the 10-monodocetaxel derivative. The derivative [4] was isolated as an off whitegum (145.1 mg, 80.86%), 24.25% calculated from starting material and itsstructure was confirmed by NMR.

Example 2 Preparation of Activated C7, C10 Docetaxel Di-acid Derivativefrom Compound [4]

The diglutaric acid derivative [4] (125.1 mg, 0.121 mmol) was dissolvedin 10 mL of dry DMSO. With stirring under nitrogenN-hydroxysulfosuccinimide sodium salt (114.7 g, 0.528 mmol, 4.4 eq) wasadded followed by EDC (102.4 mg, 0.534 mmol, 4.4 eq). The reaction wasstirred overnight at room temperature when additional EDC was added (96mg, 0.501 mmol, 4.15 eq). After 7 hours of continued stirring at roomtemperature the reaction was complete by TLC. The TLC condition wasethyl acetate: dichloromethane (3:2) with 2 drops of acetic acid.

Example 3 Preparation of Docetaxel-BSA Conjugate with Activated C7, C10Docetaxel Di-acid derivative (1:1 ratio)

To a 20 mL solution of BSA (50 mg/mL) in 50 mM phosphate buffer (50 mM,pH 7.5) with stirring on ice, was added drop wise 1.34 mL (0.016 mmol)of the activated N-hydroxysulfosuccinimide ester docetaxel derivativeprepared in example 2. The reaction mixture was allowed to stirovernight at room temperature to produce the di-acid conjugate to BSA.This conjugate was then purified by dialysis and characterized accordingto procedures described previously (Wu et. al., Bioconj. Chem., 8: pp385-390, 1997, Li et al., Bioconj. Chem., 8: pp 896-905, 1997, Salamoneet al., J. Forensic Sci. pp 821-826, 1998).

Example 4 Preparation of C7, C10 Docetaxel Di-Acid Derivative Immunogenwith BTG

To a 6.1 mL solution of BTG (32.9 mg/mL) in phosphate buffer (50 mM, pH7.5) with stirring on ice, was added drop wise 5.1 mL (0.0617 mmol) ofthe of the activated N-hydroxysulfosuccinimide ester docetaxelderivative prepared in example 2. The reaction mixture was allowed tostir overnight at room temperature to produce the di-acid conjugate toBTG. The immunogenic conjugate was then purified by dialysis andcharacterized according to procedures described previously (Wu et. al.,Bioconj. Chem., 8: pp 385-390, 1997, Li et al., Bioconj. Chem., 8: pp896-905, 1997, Salamone et al., J. Forensic Sci. pp 821-826, 1998).

Example 5 Preparation of C7, C10 Docetaxel Di-acid Derivative Immunogenwith KLH

To a 5.4 mL solution of KLH (8.9 mg/mL) in phosphate buffer (50 mM, pH7.5) with stirring on ice, was added drop wise 5.1 mL (0.0145 mmol) ofthe activated N-hydroxysulfosuccinimide ester docetaxel derivativeprepared in example 2. The reaction mixture was allowed to stirovernight at room temperature to produce the di-acid conjugate to KLH.The immunogenic conjugate was then purified by dialysis andcharacterized according to procedures described previously (Wu et. al.,Bioconj. Chem., 8: pp 385-390, 1997, Li et al., Bioconj. Chem., 8: pp896-905, 1997, Salamone et al., J. Forensic Sci. pp 821-826, 1998).

Example 6 Preparation of C7 Substituted Docetaxel Acid Derivative [6]Scheme II

To a solution of alloc-protected docetaxel, [2], (201 mg, 0.23 mmol) andDMAP (110 mg, 0.9 mmol) in DCM (6 mL) under nitrogen Et₃N, (0.9 mmol,0.13 mL) was added followed by p-nitrophenyl chloroformate (54.6 mg,0.27 mmol). The reaction mixture was stirred at room temperature for 3.5hours and then a solution of 6-amino-hexanoic acid allyl ester (52.6 mg,0.29 mmol) in DCM (2 mL) was added. The resulting mixture was stirredovernight at room temperature. DCM was removed in vacuo and the crudematerial was purified on silica gel column with EtOAc/hexanes as thegradient (Rf=0.39, 50% EtOAc/hexanes) to yield [5] (81.4 mg, 35%) as anoff-white gum.

To a solution of [5] (100 mg, 0.094 mmol) and Pd(PPh₃)₄ (15.3 mg, 0.013mmol) in DCM (6 mL) under nitrogen was added a solution of PhSiH₃ (40.8mg, 0.38 mmol) in DCM (1 mL). The resulting mixture was stirredovernight at room temperature. DCM was removed and the crude materialwas purified on a silica gel column with MeOH/DCM as the gradient(Rf=0.2, 10% MeOH/DCM) to give [6] (39.6 mg, 41%) as a tan gum and itsstructure was confirmed by NMR.

Example 7 Preparation of Activated C7 Substituted Docetaxel AcidDerivative from Compound [6]

Derivative [6] (39.6 mg, 0.042 mmol) was dissolved in 5 mL of dry DCM.With stirring under nitrogen NHS (14.5 mg, 0.126 mmol, 3.0 eq) was addedfollowed by EDC (24.0 mg, 0.126 mmol, 3.0 eq). The reaction was stirredfor 29 hours at room temperature and was then quenched by the additionof HCl (3 mL, 0.3 N) and 15 mL of DCM. The mixture was stirred for 10minutes and the organic layer was separated, dried (Na₂SO₄), filteredand the DCM was removed in vacuo to yield an off white amorphous solid.

Example 8 Preparation of Docetaxel-BSA Conjugate with Activated C7Substituted Docetaxel Acid Derivative (1:1 Ratio)

The activated ester produced in Example 6 was dissolved in 700 μL ofDMSO and 50 μL of this solution was added drop wise to 8 mL of a BSAsolution (4 mL DMSO/4 mL 50 mM phosphate, pH 7.5). The solution wasstirred for 24 hours at room temperature to produce the conjugate of BSAand the docetaxel derivative [6]. This conjugate was purified bydialysis according to procedures previously described (Wu et. al.,Bioconj. Chem., 8: pp 385-390, 1997, Li et.al., Bioconj. Chem., 8: pp896-905, 1997, Salamone et.al., J. Forensic Sci. pp 821-826, 1998).

Example 9 Preparation of C7 Substituted Docetaxel Acid derivativeImmunogen with BTG

To 6.3 mL of BTG (21.1 mg/mL) in 50 mM phosphate buffer (50 mM, pH 7.5)(DMSO) 12.6 mL was slowly added drop wise over ice. To this solution,the activated NHS ester of the C7 substituted docetaxel (derivative [6])prepared in Example 7 was added (50 μL of a 56 mg/mL in a DMSO solution)drop wise. The resulting mixture was allowed to stir overnight at roomtemperature to conjugate the BTG to the C7 docetaxel derivative. Thisimmunogneic conjugate was then purified by dialysis and characterizedaccording to procedures described previously (Wu et. al., Bioconj.Chem., 8: pp 385-390, 1997, Li et.al., Bioconj. Chem., 8: pp 896-905,1997, Salamone et.al., J. Forensic Sci. pp 821-826, 1998).

Example 10 Preparation of C7, C10 Docetaxel Di-Acid DerivativeAntibodies

Ten Female BALB/c mice were immunized i.p. with 100 μg/mouse ofDocetaxel-BTG prepared in example 4 or with Docetaxel-KLH prepared inexample 5, emulsified in Complete Freund's Adjuvant. After the initialinjection mice were boosted three times at intervals of four weeks, sixweeks and eight weeks after the preceding injection with 100 μg/mouse ofthe same immunogens emulsified in Incomplete Freund's Adjuvant. Ten daysafter the boosts test bleeds from each mouse were obtained by orbitalbleed. The anti-serum from the last test bleeds containing Docetaxelantibodies from each of the mice were evaluated by the procedures inExamples 14 and 15 to determine their reactivity to Docetaxel and theircross reactivity to 10-O-Deacytlbaccatin III and, paclitaxel [Taxol].Only the antiserum having antibodies which were selective for docetaxeland had a cross reactivity relative to docetaxel with10-O-Deacytlbaccatin III and paclitaxel of 6% or less as determined bythese screening procedures were selected.

Example 11 Preparation of C7 Substituted Docetaxel Acid derivativeAntibodies

Ten Female BALB/c mice were immunized i.p. with 100 μg/mouse ofDocetaxel-BTG prepared in example 9 emulsified in Complete Freund'sAdjuvant. After the initial injection mice were boosted twice at fourweek intervals with 100 μg/mouse of the same immunogen emulsified inIncomplete Freund's Adjuvant. Ten days after the boosts test bleeds fromeach mouse were obtained by orbital bleed. The anti-serum from the lasttest bleeds contained Docetaxel antibodies from each of the mice wereevaluated by the procedures in Examples 14 and 16 to determine theirreactivity to Docetaxel and their cross reactivity to10-O-Deacytlbaccatin III and, paclitaxel [Taxol]. Only the antiserumhaving antibodies which were selective for docetaxel and had a crossreactivity relative to docetaxel with 10-O-Deacytlbaccatin III andpaclitaxel of 6% or less as determined by these screening procedureswere selected.

Example 12 Microtiter Plate Sensitization Procedure with C7, C10Docetaxel Di-Acid Derivative—BSA Conjugate

The ELISA method for measuring Docetaxel concentrations was performed inpolystyrene microtiter plates (Nunc MaxiSorp C8 or F8 Immunomodules)optimized for protein binding and containing 96 wells per plate. Eachwell was coated with Docetaxel-BSA conjugate (prepared as in example 3)by adding 300 μL of Docetaxel-BSA conjugate at 10 μg/mL in 0.05M sodiumbicarbonate, pH=9.6, and incubating for three hours at room temperature.The wells were washed with 0.05M sodium bicarbonate, pH 9.6 and thenwere blocked with 400 μL of 5% sucrose, 0.2% sodium caseinate solutionfor 30 minutes at room temperature. After removal of the post-coatsolution the plates were dried at 37° C. overnight.

Example 13 Microtiter Plate Sensitization Procedure with C7 SubstitutedDocetaxel Acid Derivative—BSA Conjugate

The ELISA method for measuring Docetaxel concentrations was performed inpolystyrene microtiter plates (Nunc MaxiSorp C8 or F8 Immunomodules)optimized for protein binding and containing 96 wells per plate. Eachwell was coated with Docetaxel-BSA conjugate (prepared as in example 8)by adding 300 μL of Docetaxel-BSA conjugate at 10 μg/mL in 0.05M sodiumbicarbonate, pH=9.6, and incubating for three hours at room temperature.The wells were washed with 0.05M sodium bicarbonate, pH 9.6 and thenwere blocked with 400 μL of 5% sucrose, 0.2% sodium caseinate solutionfor 30 minutes at room temperature. After removal of the post-coatsolution the plates were dried at 37° C. overnight.

Example 14 Antibody Screening Procedure—Titer

The ELISA method for screening Docetaxel antibodies (produced inexamples 10 and 11) was performed with the microtiter plates that weresensitized with Docetaxel-BSA as described in examples 12 and 13. Theantibody screening assay was performed by diluting the antiseracontaining Docetaxel antibodies to 1:100, 1:1,000, 1:10,000 and1:100,000 in phosphate buffered saline containing 0.1% BSA and 0.01%thimerosal. To each well of Docetaxel-BSA sensitized wells (prepared inexamples 12 and 13) 100 μL of diluted antibody was added and incubatedfor 10 minutes at room temperature with shaking. During this incubationantibody binds to the Docetaxel-conjugate in the well. The wells of theplates were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5%Tween-80 and 0.001% Thimerosal, pH 7.8 to remove any unbound antibody.To detect the amount of Docetaxel antibody bound to the Docetaxel-BSAconjugate in the wells, 100 μL of a goat anti-mouse antibody—HRP enzymeconjugate (Jackson Immunoresearch) diluted 1/2400 in PBS with 0.1% BSA,0.05% ANS, 0.01% thimerosal, capable of binding specifically with murineimmunoglobulins and producing a colored product when incubated with asubstrate, were added to each well. After an incubation of 10 minutes atroom temperature with shaking, during which the goat anti-mouseantibody—HRP enzyme conjugate binds to Docetaxel antibodies in thewells, the plates were again washed three times to remove unbound goatanti-mouse antibody—HRP enzyme conjugate. To develop a measurable colorin the wells washing was followed by the addition of 100 μL of TMB (TMBLiquid Substrate, Sigma), a substrate for HRP, to develop color during a10 minute incubation with shaking at room temperature. Following theincubation for color development, 50 μL of stop solution (1.5% sodiumfluoride in di H₂O) was added to each well to stop the color developmentand after 10 seconds of shaking the absorbance was determined at 650 nm(Molecular Devices Plate Reader). The amount of antibody in a well wasproportional to the absorbance measured and was expressed as thedilution (titer) resulting in an absorbance of 1.5. Titers weredetermined by graphing log antibody dilution of the antibody measuredα-axis) vs. absorbance 650 nm (y-axis) and extrapolating the titer at anabsorbance of 1.5. The titer determined the concentration (dilution) ofantibody used in the indirect competitive Microtiter plate assaydescribed in examples 15 and 16.

Example 15 Indirect Competitive Microtiter Plate Immunoassay ProcedureDetermining IC50 and Cross-Reactivity for Antibodies to C7, C10Docetaxel Di-Acid Derivative Conjugate

The ELISA method for measuring Docetaxel concentrations was performedwith the microtiter plates that were sensitized with Docetaxel-BSAdescribed in example 13. Docetaxel, paclitaxel, and 10-O-deactylbaccatinIII were diluted 10 fold in PBS over a concentration range of 0.01 to10,000 ng/mL. The assay was performed by incubating 50 μL of theanalytes to be measured with 50 μL of antibody (produced in example 10with immunogen of example 5) diluted to a titer determined in example14. During the 10 minute incubation (R.T., with shaking) there is acompetition of antibody binding for the Docetaxel conjugate in the welland the analyte in solution. Following this incubation the wells of theplate were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80and 0.001% Thimerosal, pH 7.8 to remove any material that was not bound.To detect the amount of Docetaxel antibody bound to the Docetaxel-BSAconjugate in the wells, 100 μL of a goat anti-mouse antibody —HRP enzymeconjugate (Jackson Immunoresearch) diluted 1/2400 in PBS with 0.1% BSA,0.05% ANS, 0.01% thimerosal, capable of binding specifically with murineimmunoglobulins and producing a colored product when incubated with asubstrate, were added to each well. After an incubation of 10 minutes atroom temperature with shaking, during which the goat anti-mouseantibody—HRP enzyme conjugate binds to Docetaxel antibodies in thewells, the plates were again washed three times to remove unboundsecondary conjugate. To develop a measurable color in the wells washingwas followed by the addition of 100 μL of TMB (TMB Liquid Substrate,Sigma), a substrate for HRP, to develop color in a 10 minute incubationwith shaking at room temperature. Following the incubation for colordevelopment, 50 μL of stop solution (1.5% sodium fluoride in di H₂O) wasadded to each well to stop the color development and after 10 seconds ofshaking the absorbance was determined at 650 nm (Molecular Devices PlateReader). The amount of antibody in a well was proportional to theabsorbance measured and inversely proportional to the amount ofDocetaxel in the sample. The absorbance of the color in the wellscontaining analyte was compared to that with no analyte and a standardcurve was generated. The IC50 value for a given analyte was defined asthe concentration of analyte that is required to inhibit 50% of theabsorbance for the wells containing no analyte. The cross-reactivity ofa given analyte was calculated as the ratio of the IC50 for Docetaxel tothe IC50 for Paclitaxel and 10-O-Deactylbaccatin III expressed as apercent. When measured with an antibody as produced in example 10 withimmunogen of example 5 the antibodies with percent cross-reactivatesrelative to Docetaxel for Paclitaxel and 10-O-Deactylbaccatin III </=6%were obtained.

Example 16 Indirect Competitive Microtiter Plate Immunoassay ProcedureDetermining IC50 and Cross-Reactivity for Antibodies to C7 SubstitutedDocetaxel Acid Derivative Conjugate

The ELISA method for measuring Docetaxel concentrations was performedwith the microtiter plates that were sensitized with Docetaxel-BSAdescribed in examples 12 and 13. Docetaxel, Paclitaxel, and10-O-Deactylbaccatin III were diluted 10 fold in PBS over aconcentration range of 0.01 to 10,000 ng/mL. The assay was performed byincubating 50 μL of the analytes to be measured with 50 μL of antibody(produced in example 11) diluted to a titer determined in example 14.During the 10 minute incubation (R.T., with shaking) there is acompetition of antibody binding for the Docetaxel conjugate in the welland the analyte in solution. Following this incubation the wells of theplate were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80and 0.001% Thimerosal, pH 7.8 to remove any material that was not bound.To detect the amount of Docetaxel antibody bound to the Docetaxel-BSAconjugate in the wells, 100 μL of a goat anti-mouse antibody—HRP enzymeconjugate (Jackson Immunoresearch) diluted 1/2400 in PBS with 0.1% BSA,0.05% ANS, 0.01% thimerosal, capable of binding specifically with murineimmunoglobulins and producing a colored product when incubated with asubstrate, were added to each well. After an incubation of 10 minutes atroom temperature with shaking, during which the goat anti-mouseantibody—HRP enzyme conjugate binds to Docetaxel antibodies in thewells, the plates were again washed three times to remove unboundsecondary conjugate. To develop a measurable color in the wells washingwas followed by the addition of 100 μL of TMB (TMB Liquid Substrate,Sigma), a substrate for HRP, to develop color in a 10 minute incubationwith shaking at room temperature. Following the incubation for colordevelopment, 50 μL of stop solution (1.5% sodium fluoride in di H₂O) wasadded to each well to stop the color development and after 10 seconds ofshaking the absorbance was determined at 650 nm (Molecular Devices PlateReader). The amount of antibody in a well was proportional to theabsorbance measured and inversely proportional to the amount ofDocetaxel in the sample. The absorbance of the color in the wellscontaining analyte was compared to that with no analyte and a standardcurve was generated. The IC50 value for a given analyte was defined asthe concentration of analyte that is required to inhibit 50% of theabsorbance for the wells containing no analyte. The cross-reactivity ofa given analyte was calculated as the ratio of the IC50 for Docetaxel tothe IC50 for Paclitaxel, and 10-O-Deactylbaccatin III expressed as apercent. When measured with an antibody as produced in example 11 withimmunogen of example 9, on a microtiter plate prepared as in example 12the percent cross-reactivates relative to Docetaxel for Paclitaxel wasless than 2%, and for 10-O-Deacytlbaccatin III less than 0.02%. Whenmeasured with an antibody as produced in example 11 with immunogen ofexample 9, on a microtiter plate prepared as in example 13 the percentcross-reactivates relative to Docetaxel for Paclitaxel was less than 1%,and for 10-O-Deacytlbaccatin III less than 0.01% were obtained.

1. An immunoassay for detecting docetaxel in a sample comprisingproviding a mixture containing a sample, an antibody selectivelyreactive with docetaxel and not substantially cross-reactive with taxoland a conjugate of a carrier with a ligand selected from a compound ofthe formula:

wherein B is —CH₂—;

Y is an organic spacing group; X is a terminal functional group capableof binding to a carrier; and p is an integer from 0 to 1; a compound ofthe formula:

wherein B, X, Y and p are as above; a compound of the formula

wherein B, X, Y and p are as above, and mixtures thereof, causing thedocetaxel in the sample and said conjugate to bind with said antibodyand thereafter measuring the amount of said conjugate in said mixturewhich is bound or unbound to said antibody whereby the presence ofdocetaxel in the sample can be determined.
 2. The process of claim 1,wherein the sample is a human sample.
 3. The immunoassay of claim 2,wherein said antibody is generated from an immunogen comprising animmunogenic polyamine polymer linked to a ligand selected from the groupconsisting of a compound of the formula:

wherein Y and B are as above and X₂ is a functional group capable ofbinding to a polyamine polymer; a compound of the formula:

wherein B, X₂, Y and p are as above; a compound of the formula

wherein B, X₂, Y and p are as above, and mixtures thereof.
 4. Theimmunoassay of claim 2, wherein the antibody is attached to a solidsupport.
 5. The immunoassay of claim 4, wherein the solid support ismicrotitor plates.
 6. The immunoassay of claim 4, wherein the solidsupport is nanoparticles.
 7. The immunoassay of claim 6, wherein saidantibody is derived from mice, rabbits or rats.
 8. The immunoassay ofclaim 7, wherein said antibody is a monoclonal antibody.
 9. Theimmunoassay of claim 3, wherein said antibody is derived from animmunogen of an immunogenic polyamine polymer with a ligand of theformula:

wherein B is —CH₂—;

Y is an organic spacing group; X₂ is a terminal functional group capableof binding to a polyamine polymer; and p is an integer from 0 to
 1. 10.The immunoassay of claim 9, wherein said antibody is derived from mice,rabbits or rats.
 11. The immunoassay of claim 9, wherein said antibodyis a monoclonal antibody.
 12. The immunoassay of claim 3, wherein saidantibody is derived from an immunogen of an immunogenic polyaminepolymer with a ligand of the formula:

wherein B, X₂, Y and p are as above.
 13. The immunoassay of claim 12,wherein said antibody is derived from mice, rabbits or rats.
 14. Theimmunoassay of claim 13, wherein said antibody is a monoclonal antibody.15. An immunoassay of claim 13, wherein said antibody is derived from animmunogen of an immunogenic polyamine polymer and a ligand of theformula:

wherein B, X₂, Y and p are as above.
 16. The immunoassay of claim 15,wherein said antibody is derived from mice, rabbits or rats.
 17. Theimmunoassay of claim 16, wherein said antibody is a monoclonal antibody.18. An antibody which binds substantially selectively to docetaxel anddoes not substantially bind to taxol.
 19. The antibody of claim 18,wherein said antibody is generated from an immunogen comprising animmunogenic polyamine polymer linked to a ligand selected from the groupconsisting of a compound of the formula:

wherein B is —CH₂—;

Y is an organic spacing group; X₂ is a terminal functional group capableof binding to a polyamine polymer; and p is an integer from 0 to 1; acompound of the formula:

wherein B, X₂, Y and p are as above; a compound of the formula

wherein B, X₂, Y and p are as above; and mixtures thereof.
 20. Theantibody of claim 19, wherein said antibody is derived from mice,rabbits or rats.
 21. The antibody of claim 20, wherein said antibody isa monoclonal antibody.
 22. The antibody of claim 19, wherein saidantibody is derived from an immunogen of an immunogenic polyaminepolymer with a ligand of the formula:

wherein X₂, Y, B and p are as above.
 23. The antibody of claim 22,wherein said antibody is derived from mice, rabbits or rats.
 24. Theantibody of claim 23, wherein said antibody is a monoclonal antibody.25. The antibody of claim 19, wherein said antibody is derived from animmunogen of a polyamine polymer with a ligand of the formula:

wherein B, X₂, Y and p are as above.
 26. The antibody of claim 25,wherein said antibody is derived from mice, rabbits or rats.
 27. Theantibody of claim 25, wherein said antibody is a monoclonal antibody.28. The antibody of claim 25, wherein said antibody is derived frommice, rabbits or rats.
 29. The antibody of claim 19, wherein saidantibody is derived from an immunogen of a polyamine polymer and aligand of the formula:

wherein B, X₂, Y and p are as above.
 30. The antibody of claim 29,wherein said antibody is derived from mice, rabbits or rats.
 31. Theantibody of claim 30, wherein said antibody is a monoclonal antibody.32. The antibody of claim 31, wherein said antibody is derived frommice, rabbits or rats.
 33. A compound of the formula:

wherein B is —CH₂—;

Y is an organic spacing group; X is a terminal functional group capableof binding to a carrier; and p is an integer from 0 to
 1. 34. Thecompound of claim 33, wherein said carrier contains a polyamine polymer.35. The compound of claim 34, wherein p is
 0. 36. The compound of claim35, wherein X is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 37. The compound ofclaim 36, wherein X is

and R₃ is hydrogen.
 38. The compound of claim 36, wherein X is

and R₃ forms a reactive ester.
 39. The compound of claim 38, wherein theester formed is a lower alkyl ester, imidoester or amidoester.
 40. Thecompound of claim 34, wherein p is
 1. 41. The compound of claim 40,wherein X is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 42. The compound ofclaim 41, wherein Y is alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 43. A conjugate comprising a carrier linked to a ligand moiety of theformula:

wherein B is —CH₂—;

Y is an organic spacing group; X is —CH₂— or a functional linking groupcapable of linking to a carrier; and p is an integer from 0 to
 1. 44.The conjugate of claim 43, wherein the carrier comprises a polyaminepolymer.
 45. The conjugate of claim 44, wherein p is
 1. 46. Theconjugate of claim 45, wherein X′ is

wherein R₄ is oxygen or sulfur.
 47. The conjugate of claim 44, wherein pis
 1. 48. The conjugate of claim 45, wherein Y is alkylene containingfrom 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 49. The conjugate of claim 48, wherein X′ is

wherein R₄ is oxygen or sulfur.
 50. The conjugate of claim 44, whereinsaid polyamine polymer is an immunogenic polymer.
 51. A compound of theformula:

wherein B is —CH₂—;

Y is an organic spacing group; X is a functional terminal group capableof binding to a carrier; and p is an integer from 0 to
 1. 52. Thecompound of claim 51, wherein X is a functional group capable of bindingto a polyamine polymer.
 53. The compound of claim 52, wherein p is 0.54. The compound of claim 52, wherein X is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 55. The compound ofclaim 54, wherein X is

and R₃ is hydrogen.
 56. The compound of claim 54, wherein X is

and R₃ forms a reactive ester.
 57. The compound of claim 56, wherein theester formed is a lower alkyl ester, imidoester or amidoester.
 58. Thecompound of claim 51, wherein p is
 1. 59. The compound of claim 58,wherein Y is alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 60. A compound of the formula:

wherein B is —CH₂—;

Y is an organic spacing group; X is a functional terminal group capableof binding to a carrier; and p is an integer from 0 to
 1. 61. Thecompound of claim 60, wherein X is capable of binding to a polyaminepolymer.
 62. The compound of claim 61, wherein p is
 0. 63. The compoundof claim 62, wherein X is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 64. The compound ofclaim 63, wherein X is

and R₃ is hydrogen.
 65. The compound of claim 63, wherein X is

and R₃ forms a reactive ester.
 66. The compound of claim 65, wherein theester formed is a lower alkyl ester, imidoester or amidoester.
 67. Thecompound of claim 62, wherein p is
 1. 68. The compound of claim 67,wherein X is

wherein R₃ is hydrogen or taken together with its attached oxygen atomforms a reactive ester and R₄ is oxygen or sulfur.
 69. The compound ofclaim 68, wherein Y is alkylene containing from 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 70. A conjugate comprising a carrier linked to a ligand moiety of theformula:

wherein B is —CH₂—;

Y is an organic spacing group; X is —CH₂— or a functional linking groupcapable of linking to a carrier; and p is an integer from 0 to
 1. 71.The conjugate of claim 70, wherein the carrier comprises a polyaminepolymer.
 72. The conjugate of claim 71, wherein p is
 0. 73. Theconjugate of claim 72, wherein X′ is

wherein R₄ is oxygen or sulfur.
 74. The conjugate of claim 71, wherein pis
 1. 75. The conjugate of claim 74, wherein Y is alkylene containingfrom 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 76. The conjugate of claim 75, wherein X′ is

wherein R₄ is oxygen or sulfur.
 77. The conjugate of claim 71, whereinsaid polyamine polymer is an immunogenic polymer.
 78. A conjugatecomprising a carrier linked to a ligand moiety of the formula:

wherein B is —CH₂—;

Y is an organic spacing group; X¹ is —CH₂— or a functional linking groupcapable of linking to said carrier; and p is an integer from 0 to
 1. 79.The conjugate of claim 78, wherein the carrier comprises a polyaminepolymer.
 80. The compound of claim 79, wherein p is
 0. 81. The compoundof claim 80, wherein X′ is

wherein R₄ is oxygen or sulfur.
 82. The conjugate of claim 79, wherein pis
 1. 83. The conjugate of claim 82, wherein Y is alkylene containingfrom 1 to 10 carbon atoms,

wherein n and o are integers from 0 to 6, and m is an integer from 1 to6.
 84. The conjugate of claim 83, wherein X′ is

wherein R₄ is oxygen or sulfur.
 85. The conjugate of claim 79, whereinsaid polyamine polymer is an immunogenic polymer.
 86. A kit fordetermining the presence of docetaxel in a patient sample comprisingreagents packed in separate containers, one of the reagents being anantibody selectively reactive with docetaxel and not substantiallycross-reactive with taxol and the other reagent being a conjugate of thecarrier with a ligand selected from a compound of the formula:

wherein B is —CH₂—;

Y is an organic spacing group; X is a terminal functional group capableof binding to a carrier; and p is an integer from 0 to 1; a compound ofthe formula:

wherein B, X, Y and p are as above; a compound of the formula

wherein B, X, Y and p are as above; and mixtures thereof.